EP4212625A1 - Polypeptide ayant une activité d'hydroxylation de l'acide 4-aminobenzoïque et son utilisation - Google Patents

Polypeptide ayant une activité d'hydroxylation de l'acide 4-aminobenzoïque et son utilisation Download PDF

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Publication number
EP4212625A1
EP4212625A1 EP21866528.9A EP21866528A EP4212625A1 EP 4212625 A1 EP4212625 A1 EP 4212625A1 EP 21866528 A EP21866528 A EP 21866528A EP 4212625 A1 EP4212625 A1 EP 4212625A1
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Prior art keywords
position corresponding
amino acid
valine
group
amino
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English (en)
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Kyoshiro NONAKA
Fumikazu Takahashi
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Kao Corp
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Kao Corp
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/14—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with reduced flavin or flavoprotein as one donor, and incorporation of one atom of oxygen (1.14.14)
    • C12Y114/14013—4-(L-Gamma-glutamylamino)butanoyl-[BtrI acyl-carrier protein] monooxygenase (1.14.14.13)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0073—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/005—Amino acids other than alpha- or beta amino acids, e.g. gamma amino acids

Definitions

  • the present invention relates to a polypeptide having 4-aminobenzoic acid hydroxylation activity and use thereof.
  • Polybenzoxazole (PBO) is known as an engineering plastic excellent in heat resistance and mechanical strength, and used in fiber materials and insulating films of semiconductor devices and the like. (Non Patent Literature 1).
  • a benzoxazole backbone was formed by the condensation of an o-aminophenol backbone with carboxylic acid.
  • 4-amino-3-hydroxybenzoic acids (4,3-AHBAs) having these functional groups in their molecules are useful as PBO monomers.
  • 4,3-AHBAs 4-amino-3-hydroxybenzoic acids
  • Patent Literature 2 As for the production of 4,3-AHBA, for example, a synthesis method of chemically reducing a nitro aromatic compound has been known so far (Patent Literature 2).
  • a possible strategy which enables fermentative production of 4,3-AHBA by a microbial method is the hydroxylation at position 3 of 4-aminobenzoic acid (4-ABA) which can be biosynthesized within a microbe.
  • 4-ABA 4-aminobenzoic acid
  • the present invention relates to the following 1) to 7) .
  • the present invention relates to a provision of a polypeptide having excellent 4-aminobenzoic acid hydroxylation activity and a method for using the same.
  • the present inventors found that a 4-hydroxybenzoic acid hydroxylase mutant having a particular amino acid sequence has excellent 4-aminobenzoic acid hydroxylation activity and is useful in the production of 4-amino-3-hydroxybenzoic acids.
  • polypeptide having 4-aminobenzoic acid hydroxylation activity since the polypeptide having 4-aminobenzoic acid hydroxylation activity according to the present invention has excellent 4-aminobenzoic acid hydroxylation activity, use thereof enables 4-amino-3-hydroxybenzoic acids to be efficiently produced from 4-aminobenzoic acids.
  • the identity of an amino acid sequence or a nucleotide sequence is calculated by the Lipman-Pearson method ( Science, 1985, 227: 1435-1441 ). Specifically, the identity is calculated by analysis with a unit size to compare (ktup) set to 2 using the homology analysis (search homology) program of genetic information processing software GENETYX Ver. 12.
  • the "position corresponding" on an amino acid sequence or a nucleotide sequence can be determined by aligning a sequence of interest and a reference sequence (e.g., the amino acid sequence represented by SEQ ID NO: 2) so as to provide the maximum homology.
  • the alignment of amino acid sequences or nucleotide sequences can be carried out using an algorithm known in the art, and the procedures thereof are known to those skilled in the art. The alignment can be performed, for example, by using default settings of Clustal W multiple alignment program ( Thompson, J.D. et al., 1994, Nucleic Acids Res. 22: 4673-4680 ).
  • Clustal W2 or Clustal omega a modified version of Clustal W, may be used.
  • Clustal W, Clustal W2 and Clustal omega are available on, for example, the website of European Bioinformatics Institute (EBI [www.ebi.ac.uk/index.html]) or the DNA Databank of Japan (DDBJ [www.ddbj.nig.ac.jp/searches-j.html]) run by National Institute of Genetics.
  • EBI European Bioinformatics Institute
  • DDBJ DNA Databank of Japan
  • a position of the sequence of interest aligned to an arbitrary position of the reference sequence by the alignment mentioned above is regarded as a "position corresponding" to the arbitrary position.
  • Those skilled in the art can further finely adjust the alignment of amino acid sequences obtained as described above for optimization. Such optimum alignment is preferably determined in consideration of the similarity between the amino acid sequences, the frequency of a gap to be inserted and the like.
  • the similarity between the amino acid sequences refers the ratio (%) of the number of positions at which identical or similar amino acid residues are present in both the sequences to the number of full-length amino acid residues when these two amino acid sequences are aligned.
  • the similar amino acid residues mean amino acid residues which are similar in property to each other in terms of polarity or electric charge and cause so-called conservative substitution, among 20 amino acids constituting a protein. Groups consisting of such similar amino acid residues are well known to those skilled in the art.
  • Examples thereof include, but are not limited to: arginine and lysine or glutamine; glutamic acid and aspartic acid or glutamine; serine and threonine or alanine; glutamine and asparagine or arginine; and leucine and isoleucine.
  • amino acid residue means any of 20 amino acid residues constituting a protein, i.e., alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y) and valine (Val or V).
  • alanine Al or A
  • arginine Arg or R
  • asparagine Asn or N
  • the "operable linkage" of a gene to a control region such as a promoter means that the gene is linked to the control region such that the gene is expressible under the control of the control region.
  • the procedures of the "operable linkage" of a gene to a control region are well known to those skilled in the art.
  • upstream and downstream in relation to a gene refer to upstream and downstream in the direction of transcription of the gene.
  • the phrase “gene located downstream of a promoter” means that the gene resides on the 3' side of the promoter in a DNA sense strand
  • the phrase “upstream of a gene” means a region on the 5' side of the gene in a DNA sense strand.
  • the term "original” which is used for a function, property, or trait of a cell is used for indicating that the function, the property, or the trait is indigenous to the cell.
  • the term “foreign” is used for indicating that the function, the property, or the trait is introduced ab extra, not indigenous to the cell.
  • a “foreign" gene or polynucleotide is a gene or a polynucleotide introduced ab extra to a cell.
  • the foreign gene or polynucleotide may be derived from an organism of the same species as that of the cell in to which the gene or polynucleotide is introduced or may be derived from an organism of different species therefrom (i.e., a heterologous gene or polynucleotide).
  • polypeptide having 4-aminobenzoic acid hydroxylation activity is a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence having at least 90% identity thereto, and having an amino acid residue at position 39, 43, 83, 106, 180, 266, 346, 363 or 371 of the amino acid sequence represented by SEQ ID NO: 2 or a position corresponding to the position 39, 43, 83, 106, 180, 266, 346, 363 or 371 being the following amino acid:
  • the polypeptide is a mutant polypeptide having 4-aminobenzoic acid hydroxylation activity, which consists of a polypeptide serving as a reference, i.e., a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence having at least 90% identity thereto, and in which an amino acid residue at position 39, 43, 83, 106, 180, 266, 346, 363 or 371 of the amino acid sequence represented by SEQ ID NO: 2 or a position corresponding to the position 39, 43, 83, 106, 180, 266, 346, 363 or 371 is substituted with any of the amino acids (a) to (i).
  • the "4-aminobenzoic acid hydroxylation activity” means activity of catalyzing the hydroxylation of 4-aminobenzoic acids, preferably activity of catalyzing the hydroxylation at position 3 of 4-aminobenzoic acids.
  • the 4-aminobenzoic acid hydroxylation activity can be determined, as shown in Examples mentioned later, by culturing a microorganism producing the polypeptide of the present invention, and measuring the amount of 4-amino-3-hydroxybenzoic acid produced by HPLC or the like.
  • the polypeptide of the present invention can be produced by substituting an amino acid residue at position 39, 43, 83, 106, 180, 266, 346, 363 or 371 of the amino acid sequence represented by SEQ ID NO: 2 or a position corresponding to the position 39, 43, 83, 106, 180, 266, 346, 363 or 371 with the following amino acid, in a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence having at least 90% identity thereto, and having 4-aminobenzoic acid hydroxylation activity:
  • polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence having at least 90% identity thereto, and having 4-aminobenzoic acid hydroxylation activity is a "parent" polypeptide of the polypeptide of the present invention.
  • the "parent" polypeptide refers to a reference polypeptide which becomes the polypeptide of the present invention by introducing a predetermined mutation to the amino acid residue.
  • HFM122 a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 (NCBI Reference Sequence: WP_010920262.1), is known as 4-hydroxybenzoate 3-monooxygenase (EC1.14.13.2).
  • the 4-hydroxybenzoate 3-monooxygenase is an enzyme having catalytic activity of accelerating any one or both of reaction to produce protocatechuic acid by hydroxylating position 3 of 4-hydroxybenzoic acid and inverse reaction thereof, and is an enzyme which catalyzes the hydroxylation of 4-hydroxybenzoic acids (4-hydroxybenzoic acid hydroxylase).
  • This HFM122 was found to have 4-aminobenzoic acid hydroxylation activity by the present applicant ( Japanese Patent Application No. 2018-171849 ).
  • polypeptide consisting of an amino acid sequence having at least 90% identity to the amino acid sequence represented by SEQ ID NO: 2, and having 4-aminobenzoic acid hydroxylation activity
  • polypeptide which consists of an amino acid sequence having at least 90% identity, specifically, 90% or higher, preferably 95% or higher, more preferably 96% or higher, further preferably 97% or higher, further preferably 98% or higher, further preferably 99% or higher identity to the amino acid sequence represented by SEQ ID NO: 2, and has 4-aminobenzoic acid hydroxylation activity.
  • the parent polypeptide preferably has a valine residue at position 39 of the amino acid sequence represented by SEQ ID NO: 2 or a position corresponding thereto, preferably has a valine residue at position 43 thereof or a position corresponding thereto, preferably has a methionine residue at position 83 thereof or a position corresponding thereto, preferably has a methionine residue at position 106 thereof or a position corresponding thereto, preferably has a valine residue at position 180 thereof or a position corresponding thereto, preferably has an alanine residue at position 266 thereof or a position corresponding thereto, preferably has a phenylalanine residue at position 346 thereof or a position corresponding thereto, preferably has a methionine residue at position 363 thereof or a position corresponding thereto, or preferably has an isoleucine residue at position 371 thereof or a position corresponding thereto.
  • valine at the position 39 or the position corresponding thereto with cysteine it is more preferred to substitute valine at the position 39 or the position corresponding thereto with cysteine, to substitute valine at the position 43 or the position corresponding thereto with leucine or histidine, to substitute methionine at the position 83 or the position corresponding thereto with valine, glycine or phenylalanine, to substitute methionine at the position 106 or the position corresponding thereto with alanine, cysteine, isoleucine or threonine, to substitute valine at the position 180 or the position corresponding thereto with alanine, to substitute alanine at the position 266 or the position corresponding thereto with valine or phenylalanine, to substitute phenylalanine at the position 346 or the position corresponding thereto with leucine, to substitute methionine at the position 363 or the position corresponding thereto with valine or leucine, or to substitute isoleucine at the position 371 or
  • valine at the position 43 or the position corresponding thereto with histidine to substitute methionine at the position 106 or the position corresponding thereto with alanine, isoleucine or threonine, to substitute valine at the position 180 or the position corresponding thereto with alanine, to substitute alanine at the position 266 or the position corresponding thereto with valine, to substitute phenylalanine at the position 346 or the position corresponding thereto with leucine, to substitute methionine at the position 363 or the position corresponding thereto with leucine, or to substitute isoleucine at the position 371 or the position corresponding thereto with phenylalanine.
  • any of various mutagenesis techniques known in the art can be used as an approach of mutating the amino acid residue of the parent polypeptide.
  • a nucleotide sequence encoding the amino acid residue to be mutated in a polynucleotide encoding the amino acid sequence of the parent polypeptide (hereinafter, also referred to as a parent gene) can be mutated to a nucleotide sequence encoding a mutated amino acid residue to obtain a polynucleotide encoding the polypeptide of the present invention.
  • the introduction of the mutation of interest to the parent gene can be basically performed by use of any of various site-directed mutagenesis methods well known to those skilled in the art.
  • the site-directed mutagenesis method can be performed by, for example, any approach such as inverse PCR or annealing.
  • a commercially available kit for site-directed mutagenesis e.g., QuikChange II Site-Directed Mutagenesis Kit or QuikChange Multi Site-Directed Mutagenesis Kit from Agilent Technologies, Inc. may be used.
  • the site-directed mutagenesis of the parent gene can be performed most generally using primers for mutations containing a nucleotide mutation to be introduced.
  • the primers for mutations can be designed so as to contain a nucleotide sequence which is annealed to a region containing a nucleotide sequence encoding the amino acid residue to be mutated in the parent gene, and has a nucleotide sequence (codon) encoding the mutated amino acid residue instead of the nucleotide sequence (codon) encoding the amino acid residue to be mutated.
  • the nucleotide sequences (codons) encoding the unmutated or mutated amino acid residues can be appropriately recognized and selected by those skilled in the art on the basis of a usual textbook or the like.
  • the site-directed mutagenesis may employ a method of linking DNA fragments respectively amplified from the upstream and downstream sides of a mutation site by separately using two complementary primers containing a nucleotide mutation to be introduced, into one fragment by SOE (splicing by overlap extension)-PCR ( Gene, 1989, 77 (1): p. 61-68 ).
  • Template DNA containing the parent gene can be prepared by extracting genomic DNA by a routine method from a microorganism producing the 4-hydroxybenzoic acid hydroxylase mentioned above, or by extracting RNA therefrom and synthesizing cDNA by reverse transcription.
  • a corresponding nucleotide sequence may be chemically synthesized on the basis of the amino acid sequence of the parent polypeptide, and used as template DNA.
  • DNA sequences containing nucleotide sequences encoding HFM122 already mentioned as polypeptides having 4-aminobenzoic acid hydroxylation activity are shown in SEQ ID NO: 1.
  • the primers for mutations can be prepared by a well-known oligonucleotide synthesis method such as phosphoramidite method ( Nucleic Acids R4esearch, 1989, 17: 7059-7071 ). Such primer synthesis may be carried out using, for example, a commercially available oligonucleotide synthesis apparatus (manufactured by Applied Biosystems Inc. (ABI) or the like.).
  • the polynucleotide encoding the polypeptide of the present invention having the mutation of interest can be obtained by the site-directed mutagenesis as described above with the parent gene as template DNA using a primer set containing the primers for mutations.
  • the polynucleotide encoding the polypeptide of the present invention may contain single-stranded or doublestranded DNA, cDNA, RNA or other artificial nucleic acids.
  • the DNA, the cDNA and the RNA may be chemically synthesized.
  • the polynucleotide may also contain an open reading frame (ORF) as well as the nucleotide sequence of an untranslated region (UTR).
  • ORF open reading frame
  • the polynucleotide may be codon-optimized for the species of the transformant for mutant polypeptide production of the present invention. Information on codons which are used by various organisms is available from Codon Usage Database ([www.kazusa.or.jp/codon/]).
  • the obtained polynucleotide encoding the polypeptide of the present invention can be inserted to a vector.
  • the vector containing the polynucleotide is an expression vector.
  • the vector is an expression vector which can introduce the polynucleotide encoding the polypeptide of the present invention into a host microorganism and enables the polynucleotide to be expressed within the host microorganism.
  • the vector contains the polynucleotide encoding the polypeptide of the present invention, and a control region operably linked thereto.
  • the vector may be a vector capable of proliferating and replicating autonomously outside the chromosome (i.e., in a plasmid), or may be a vector which is integrated into the chromosome.
  • the vector examples include pBluescript II SK(-) (Stratagene California), pUC series vectors such as pUC18/19 and pUC118/119 (Takara Bio Inc.), pET series vectors (Takara Bio Inc.), pGEX series vectors (GE Healthcare Japan Corp.), pCold series vectors (Takara Bio Inc.), pHY300PLK (Takara Bio Inc.), pUB110 ( Mckenzie, T.
  • Plasmid 15 (2): 93-103 Plasmid 15 (2): 93-103
  • pBR322 Tropa Bio Inc.
  • pRS403 Stratagene California
  • pMW218/219 Nippon Gene Co., Ltd.
  • pRI series vectors such as pRI909/910 (Takara Bio Inc.), pBI series vectors (Clontech Laboratories, Inc.), IN3 series vectors (Inplanta Innovations Inc.), pPTR1/2 (Takara Bio Inc.), pDJB2 ( D.J.
  • the polynucleotide encoding the polypeptide of the present invention may be constructed as a DNA fragment containing the same.
  • the DNA fragment include PCR-amplified DNA fragments and restriction enzyme-cleaved DNA fragments.
  • the DNA fragment can be an expression cassette containing the polynucleotide encoding the polypeptide of the present invention, and a control region operably linked thereto.
  • the control region contained in the vector or the DNA fragment is a sequence for allowing the polynucleotide encoding the polypeptide of the present invention to be expressed within a host cell into which the vector or the DNA fragment is introduced. Examples thereof include expression regulation regions such as promoters and terminators, and replication origins.
  • the type of the control region can be appropriately selected according to the type of the host microorganism into which the vector or the DNA fragment is introduced. If necessary, the vector or the DNA fragment may further have a selective marker such as an antibiotic resistance gene or an amino acid synthesis-related gene (e.g., a resistance gene for a drug such as ampicillin, neomycin, kanamycin, or chloramphenicol).
  • the vector or the DNA fragment may contain a polynucleotide sequence encoding a polypeptide necessary for biosynthesizing 4-aminobenzoic acids.
  • polypeptide necessary for biosynthesizing 4-aminobenzoic acids include 4-amino-4-deoxychorismate synthase (pabAB) and 4-amino-4-deoxychorismate lyase (pabC).
  • the linkage of the polynucleotide encoding the polypeptide of the present invention to the control region or the marker gene sequence can be performed by a method such as the SOE-PCR mentioned above. Procedures of introducing the gene sequence to the vector are well known in the art.
  • the type of the control region such as a promoter region, a terminator, or a secretion signal region is not particularly limited, and a promoter or a secretion signal sequence usually used can be appropriately selected and used according to the recipient host.
  • control region include, but are not particularly limited to, strong control regions which can enhance expression as compared with a wild type, for example, high-expression promoters known in the art such as T7 promoter, lac promoter, tac promoter, and trp promoter.
  • the transformed cell of the present invention can be obtained by introducing the vector containing the polynucleotide encoding the polypeptide of the present invention into a host, or by introducing the DNA fragment containing the polynucleotide encoding the polypeptide of the present invention into the genome of a host.
  • the transformed cell is a cell into which the polynucleotide encoding the polypeptide of the present invention is introduced to express it, and can be a cell having the enhanced expression of the polynucleotide, and by extension, a cell having the enhanced expression of the polypeptide of the present invention.
  • any of cells of a fungi, a yeast, actinomycete, E . coli, Bacillus subtilis, or the like may be used as a host cell, and E. coli or actinomycete is preferred.
  • the actinomycete include a bacterium of the genus Corynebacterium, a bacterium of the genus Mycobacterium, a bacterium of the genus Rhodococcus, a bacterium of the genus Streptomyces, and a bacterium of the genus Propionibacterium.
  • a bacterium of the genus Corynebacterium is preferred, and Corynebacterium glutamicum is more preferred.
  • a microorganism which can supply 4-aminobenzoic acids serving as substrates in the biosynthesis of 4-amino-3-hydroxybenzoic acids is preferred, and a microorganism having enhanced ability to supply 4-aminobenzoic acids is more preferred.
  • Examples of the method for enhancing the ability of the microorganism to supply 4-aminobenzoic acids include a method of introducing, into the microorganism, a vector containing a polynucleotide encoding a polypeptide necessary for biosynthesizing 4-aminobenzoic acids, and a control region operably linked thereto, and a method of substituting the control region of a polynucleotide encoding a polypeptide necessary for biosynthesizing 4-aminobenzoic acids, which is originally carried by the microorganism, with a strong-expression promoter.
  • Examples of the method for introducing the vector or the DNA fragment into the host include, for example, electroporation, transformation, transfection, conjugation, protoplast method, particle gun method, or Agrobacterium method.
  • Examples of the method for introducing the polynucleotide into the host include, but are not particularly limited to, a double crossover method using the DNA fragment containing the polynucleotide.
  • the DNA fragment may be introduced into the downstream of a promoter sequence of a gene having a large expression level in the host cell mentioned above, or a fragment of the DNA fragment operably linked to the control region mentioned above may be prepared in advance, and the linked fragment can be introduced into the genome of the host.
  • the DNA fragment may be further linked in advance to a marker (drug resistance gene, auxotrophic complementary gene and the like) for selecting a cell into which the polynucleotide of the present invention is introduced properly.
  • the transformed cell into which the vector or the DNA fragment of interest is introduced can be selected by exploiting the selective marker.
  • the selective marker is, for example, an antibiotic resistance gene
  • the transformed cell into which the vector or the DNA fragment of interest is been introduced can be selected by culture in a culture medium supplemented with the antibiotic.
  • the selective marker is, for example, an amino acid synthesis-related gene
  • the transformed cell into which the vector or the DNA fragment of interest is introduced can be selected by using the presence or absence of the amino acid auxotrophy as an index after introduction of gene into a host microorganism requiring the amino acid.
  • the introduction of the vector or the DNA fragment of interest may be confirmed by examining the DNA sequence of the transformed cell by PCR or the like.
  • the transformed cell obtained above is cultured in a proper culture medium so that the polynucleotide introduced into the cell is expressed to produce the polypeptide of the present invention.
  • the transformed cell is capable of serving as a bacterium producing a polypeptide having 4-aminobenzoic acid hydroxylation activity.
  • the productivity of 4-amino-3-hydroxybenzoic acid is improved as compared with the case of using a transformed cell producing the parent polypeptide.
  • the mutation to substitute an amino acid residue at position 39, 43, 83, 106, 180, 266, 346, 363 or 371 of the amino acid sequence represented by SEQ ID NO: 2 or a position corresponding to the position 39, 43, 83, 106, 180, 266, 346, 363 or 371 with the following amino acid is useful in improvement in 4-aminobenzoic acid hydroxylation activity and also useful in improvement in the productivity of 4-amino-3-hydroxybenzoic acids:
  • the transformed cell of the present invention serves as a bacterium producing a polypeptide having improved 4-aminobenzoic acid hydroxylation activity and serves as a useful strain producing 4-amino-3-hydroxybenzoic acids.
  • the method for producing 4-amino-3-hydroxybenzoic acids of the present invention includes a step of culturing the transformed cell of the present invention, and 4-amino-3-hydroxybenzoic acids can be obtained by collecting the 4-amino-3-hydroxybenzoic acids from a culture medium.
  • 4-amino-3-hydroxybenzoic acids include 4-amino-3-hydroxybenzoic acid derivatives of the following formula (1) : wherein R 1 represents a hydrogen atom, a hydroxy group (-OH), a methoxy group (-OCH 3 ), an amino group (-NH 2 ), a fluorine atom (-F), a chlorine atom (-Cl), a bromine atom (-Br), an iodine atom (-I), a carboxy group (-COOH), a methyl group (-CH 3 ), or an ethyl group (-CH 2 CH 3 ), R 2 represents a hydrogen atom, a hydroxy group (-OH), a methoxy group (-OCH 3 ), an amino group (-NH 2 ), a fluorine atom (-F), a chlorine atom (-Cl), a bromine atom (-Br), an iodine atom (-I), a carboxy group (--
  • the functional group R 1 is preferably a hydrogen atom, a hydroxy group (-OH), a methoxy group (-OCH 3 ), a fluorine atom (-F) or a methyl group (-CH 3 ).
  • the functional group R 2 is preferably a hydrogen atom, a hydroxy group (-OH), a methoxy group (-OCH 3 ), a fluorine atom (-F) or a methyl group (-CH 3 ).
  • both R 1 and R 2 are hydrogen atoms.
  • Both X 1 and X 2 may be hydroxy groups, and either one of X 1 or X 2 is preferably a hydroxy group.
  • the culture medium can contain, if necessary, 4-aminobenzoic acids serving as substrates in the biosynthesis of 4-amino-3-hydroxybenzoic acids.
  • examples of the 4-aminobenzoic acids include 4-aminobenzoic acid derivatives of the following formula (2): wherein R 1 and R 2 are as defined above.
  • any of a natural culture medium and a synthetic culture medium may be used as the culture medium for the culture of the transformed cell as long as the culture medium contains a carbon source, a nitrogen source, inorganic salts and the like and permits efficient culture of the transformed cell of the present invention.
  • a carbon source such as glucose
  • polyols such as glycerin
  • alcohols such as ethanol
  • organic acids such as pyruvic acid, succinic acid or citric acid
  • peptone, meat extracts, yeast extracts, casein hydrolysates, alkali extracts of soybean meal, alkylamines such as methylamine, or ammonia or its salt can be used as nitrogen sources.
  • phosphate, carbonate, sulfate, salts of magnesium, calcium, potassium, iron, manganese, zinc, or the like, a particular amino acid, a particular vitamin, an antifoaming agent or the like may be used, if necessary.
  • the culture can usually be performed, if necessary, with stirring or shaking, at 10°C to 40°C for 6 hours to 72 hours, preferably for 9 hours to 60 hours, more preferably for 12 hours to 48 hours.
  • an antibiotic such as ampicillin or kanamycin may be added, if necessary, to the culture medium.
  • the methods for collecting and purifying 4-amino-3-hydroxybenzoic acids from the cultures are not particularly limited. Specifically, the collection and the purification can be carried out by combining a well-known ion-exchange resin method, precipitation method, crystallization method, recrystallization method, concentration method and other methods. For example, after removal of bacterial cells by centrifugation or the like, ionic substances are removed with cation- and anion-exchange resins, and the resultant can be concentrated to obtain 4-amino-3-hydroxybenzoic acids.
  • the 4-amino-3-hydroxybenzoic acids accumulated in the cultures may be used directly without being isolated.
  • the present invention also encompasses the following substances, production methods, use, methods and the like as exemplary embodiments. However, the present invention is not limited by these embodiments.
  • PCR was performed using PrimeSTAR Max DNA Polymerase (Takara Bio Inc.) unless otherwise specified.
  • a DNA fragment containing genes encoding 4-amino-4-deoxychorismate synthase and 4-amino-4-deoxychorismate lyase was amplified by PCR using the genome extracted from a Corynebacterium glutamicum ATCC13032 strain by a routine method as a template and using primers GN14_127 (SEQ ID NO: 3, TATTAATTAAATGCGCGTTTTAATTATTGATAATTATGATTC) and GN14_133 (SEQ ID NO: 4, TTGCGGCCGCTTGTTTAAACCTCCTTACAGAAAAATGGTTGGGCG).
  • This fragment was inserted between the PacI site and the NotI site of a plasmid pECsf_gapS (see Japanese Patent Application No. 2015-25491 ) to obtain a plasmid pECsf_gapS_pabABC.
  • a DNA fragment for a vector was synthesized by PCR using the plasmid pECsf_gapS_pabABC obtained above as a template and using primers pabABCcory vec R (SEQ ID NO: 5, AAATTTAAACCTCCTTTACAGAAAAATGGTTGG) and pabABCcory vec F (SEQ ID NO: 6, GGAGGTTTAAACAAGCGGCCGCGATATC).
  • a plasmid containing a gene (SEQ ID NO: 1) encoding a polypeptide HFM122 having 4-aminobenzoic acid hydroxylation activity was prepared by artificial gene synthesis, and a DNA fragment for an insert was synthesized by PCR using this plasmid as a template and using primers pECsfD HFM122 F (SEQ ID NO: 7, AGGAGGTTTAAATTTATGCGCACTCAGGTGGCTAT) and pECsfD HFM122 R (SEQ ID NO: 8, CTTGTTTAAACCTCCTTATACGAGTGGCAGTCCTA). These PCR products were treated with DpnI (Takara Bio Inc.).
  • the respective DNA fragments were purified using NucleoSpin Gel and PCR Clean-up (Takara Bio Inc.) and ligated using In-Fusion HD Cloning Kit (Takara Bio Inc.) to construct a plasmid pECsf_gapS_pabABC_HFM122.
  • the ECOS Competent E. coli DH5 ⁇ strain was transformed with the obtained plasmid solution.
  • the cell solution was spread over LBKm agar medium (1% Bacto Trypton, 0.5% yeast extract, 1% NaCl, 50 ⁇ g/mL kanamycin sulfate, 1.5% agar) and then left standing overnight at 37°C.
  • the obtained colonies were subjected to PCR reaction using Sapphire Amp (Takara Bio Inc.) and primers pabABC+pobA for CPCR F (SEQ ID NO: 9, GCTATCAAAACATTCGGCACATTGGTTTTCC) and pabABC+pobA for CPCR R (SEQ ID NO: 10, GGAAGATGCGTGATCTGATCCTTCAACTC) to select a transformant confirmed to have the introduced DNA fragment of interest.
  • the obtained transformant was inoculated to 2 mL of LBKm liquid medium (1% Bacto Trypton, 0.5% yeast extract, 1% NaCl, 50 ⁇ g/mL kanamycin sulfate) and cultured overnight at 37°C.
  • a plasmid was purified from this culture solution using NucleoSpin Plasmid EasyPure (Takara Bio Inc.).
  • a DNA fragment for a vector was synthesized by PCR using the plasmid pECsf_gapS_pabABC_HFM122 obtained above as a template and using primers pabC last R (SEQ ID NO: 11, TTACAGAAAAATGGTTGGGCGCAA) and HFM122 F (SEQ ID NO: 12, ATGCGCACTCAGGTGGCTATCG).
  • tu promoter tuf gene (cg0587) promoter (hereinafter, referred to as tu promoter) carried by a Corynebacterium glutamicum ATCC13032 strain was prepared by artificial gene synthesis, and a DNA fragment for an insert was synth
  • PCR products were treated with DpnI (Takara Bio Inc.). Then, the respective DNA fragments were purified using NucleoSpin Gel and PCR Clean-up (Takara Bio Inc.) and ligated using In-Fusion HD Cloning Kit (Takara Bio Inc.) to construct a plasmid pECsf_gapS_pabABC_tuD_HFM122.
  • the ECOS Competent E. coli DH5 ⁇ strain was transformed with the obtained plasmid solution. The cell solution was spread over LBKm agar medium and then left standing overnight at 37°C.
  • the obtained colonies were subjected to PCR reaction using Sapphire Amp (Takara Bio Inc.) and primers Ptu seq 1 (SEQ ID NO: 16, GCTTGTTAGATATCTTGAAATCGGCTTTC) and pabABC+pobA for CPCR R (SEQ ID NO: 10, GGAAGATGCGTGATCTGATCCTTCAACTC) to select a transformant confirmed to have the introduced DNA fragment of interest.
  • the obtained transformant was inoculated to 2 mL of LBKm liquid medium and cultured overnight at 37°C. A plasmid was purified from this culture solution using NucleoSpin Plasmid EasyPure (Takara Bio Inc.).
  • the genes encoding 4-amino-4-deoxychorismate synthase and 4-amino-4-deoxychorismate lyase were linked under the control of gap promoter, and the gene encoding wild-type HFM122 was further linked under the control of the tu promoter.
  • the preparation of a plasmid containing a gene encoding a mutant enzyme will be given below by taking, as an example, the preparation of a plasmid containing a gene encoding a mutant enzyme in which valine at position 39 of HFM122 was substituted with cysteine.
  • a plasmid pECsf_gapS_pabABC_tuD_HFM122_V39C was constructed by PCR using a plasmid pECsf_gapS_pabABC_tuD_HFM122 as a template and using complementary primers HFM122 V39C F (SEQ ID NO: 17, GCTTATTGTGAAGGCCGAGTTCGGGCT) and HFM122 V39C R (SEQ ID NO: 18, GCCTTCACAATAAGCACGGTCTTTGCG).
  • HFM122 V39C F SEQ ID NO: 17, GCTTATTGTGAAGGCCGAGTTCGGGCT
  • HFM122 V39C R SEQ ID NO: 18, GCCTTCACAATAAGCACGGTCTTTGCG.
  • the PCR product was treated with DpnI (Takara Bio Inc.).
  • the ECOS Competent E. coli DH5 ⁇ strain was transformed with the solution thus treated.
  • the cell solution was spread over LBKm agar medium and then left standing overnight at 37°C. The obtained colonies were selected as a transformant.
  • the transformant was inoculated to 2 mL of LBKm liquid medium and cultured overnight at 37°C. A plasmid was purified from this culture solution using NucleoSpin Plasmid EasyPure (Takara Bio Inc.).
  • a Corynebacterium glutamicum DRHG145 strain (see Japanese Patent Application No. 2014-523757 ) was transformed with each plasmid obtained above by electroporation (Bio-Rad Laboratories, Inc.). The obtained transformed cell solution was spread over LBKm agar medium and then left standing at 30°C for 2 days. The obtained colonies were used as a transformant.
  • Each transformant obtained above was inoculated to 600 ⁇ L of CGXII medium (containing 50 ⁇ g/mL kanamycin sulfate) shown in Table 2, and cultured at 30°C for approximately 48 hours. Then, bacterial cells were removed by centrifugation to obtain a culture supernatant. The concentration of 4-amino-3-hydroxybenzoic acid in the obtained culture supernatant was quantified in accordance with the method of Reference Example 1. The rate of improvement in the ability to produce 4-amino-3-hydroxybenzoic acid was calculated in accordance to the equation given below.
  • WT represents a “transformant into which the plasmid containing the gene encoding the wild-type enzyme is introduced”
  • MT represents a “transformant into which the plasmid containing the gene encoding the mutant enzyme is introduced”.
  • Rate of improvement in production ability Ability of MT to produce 4 ⁇ amino ⁇ 3 ⁇ hydroxybenzoic acid / Ability of WT to produce 4 ⁇ amino ⁇ 3 ⁇ hydroxybenzoic acid
  • CGXII medium composition (per L) Glucose 50g (NH 4 ) 2 SO 4 20g Urea 5g KH 2 PO 4 1g K 2 HPO 4 1g MgSO 4 ⁇ 7H 2 O 0.25g CaCl 2 ⁇ 2H 2 O 10mg FeSO 4 ⁇ 7H 2 O 10mg MnSO 4 ⁇ 5H 2 O 10mg ZnSO 4 ⁇ 7H 2 O 1mg CuSO 4 ⁇ 5H 2 O 0.2mg NiCl 2 ⁇ 6H 2 O 0.02mg Biotin (pH7) 0.2mg Tryptone 50g
  • 4-Amino-3-hydroxybenzoic acid was quantified by HPLC.
  • a reaction solution to be subjected to HPLC analysis was appropriately diluted with 0.1% phosphoric acid. Then, insoluble matter was removed using AcroPrep 96-well filter plates (0.2 ⁇ m GHP membrane, Nihon Pall Ltd.).
  • the HPLC apparatus used was Chromaster (Hitachi High-Tech Science Corp.).
  • the analytical column used was L-column CDS (4.6 mm I.D. ⁇ 150 mm, Chemicals Evaluation and Research Institute, Japan).
  • Eluent A was a 0.1% phosphoric acid solution of 0.1 M potassium dihydrogen phosphate
  • eluent B was 70% methanol. Gradient elution was performed under conditions involving a flow rate of 1.0 mL/min and a column temperature of 40°C.
  • a UV detector (detection wavelength: 280 nm) was used for the detection of 4-amino-3-hydroxybenzoic acid.
  • a concentration calibration curve was prepared using a standard sample [4-amino-3-hydroxybenzoic acid (distributor code A1194, Tokyo Chemical Industry Co., Ltd.)]. 4-Amino-3-hydroxybenzoic acid was quantified on the basis of the concentration calibration curve.

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